Endothelial cell growth medium 2 egm2

Human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) were purchased from PromoCell Cells (Heidelberg, Germany) and were subsequently cultured in PromoCell’s Endothelial Cell Growth Medium 2 (EGM-2) containing 2% foetal bovine serum (FBS), human epidermal growth factor (EGF), human basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), human vascular endothelial growth factor (VEGF), ascorbic acid and heparin. Endothelial Cell Growth Medium-2 as well as their respective bullet kits were purchased from PromoCell. Trypsin 0.005%/EDTA 0.01% solution, Dulbecco A Phosphate Buffered Saline (PBS), 1-StepTM NBT/BCIP (Pierce Protein Research) were purchased from Thermo Scientific, Loughborough, United Kingdom. Foetal bovine serum (FBS), dimethyl sulfoxide (DMSO), paraformaldehyde, Triton^® X-100, rabbit anti-human von Willebrand factor antibody (F-3520), and mouse anti-rabbit IgG-alkaline phosphatase (A9919) were purchased from Sigma-Aldrich, Gillingham, Dorset, United Kingdom. Human recombinant VEGF and precast gels were purchased from Invitrogen Life Technologies, Paisley, United Kingdom. PTK787 (vatalanib dihydrochloride) was purchased from MedChem Express, Monmouth Junction, United States. Pronase E were purchased from Solarbio, Shanghai, China.

Immortalized MDFs and LFs, Platinum-E, and human embryotic kidney 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin under 10% CO₂. LECs were seeded on 0.1% gelatine-coated wells and cultured in a 1:1 mixture of Endothelial Cell Growth Medium 2 (EGM2) (PromoCell) and fully supplemented DMEM (Merck) [containing 20% fetal calf serum, glucose (4 g/liter), 2 mM glutamine, 1% penicillin (100 U/ml)–streptomycin (100 μg/ml), sodium pyruvate 1% (1 mM), Hepes (20 mM), and 1% nonessential amino acids]. Vero E6 cells were supplied by J. Vergara from the Centro de Investigación en Sanidad Animal IRTA-CReSA (Barcelona, Spain). Vero E6 cells were grown in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 2 mM GlutaMAX (Gibco), penicillin (100 U/ml; Sigma-Aldrich), streptomycin (100 μg/ml; Sigma-Aldrich), amphotericin B (0.25 μg/ml; Sigma-Aldrich), 1% nonessential amino acids (Gibco), and 25 mM Hepes (Biowest).

Human fibrosarcoma HT1080 cells were obtained from the Korean Cell Line Bank (KCLB, No. 10121) and maintained in RPMI1640 media (Hyclone Laboratories, Logan, UT, United States) with 10% fetal bovine serum (FBS, Hyclone Laboratories) and penicillin/streptomycin (Cellgro, Manassas, VA, United States) at 37°C in a humidified 5% CO₂ incubator. Human umbilical vein endothelial cells (HUVECs) were obtained from Innopharmascreen (Asan, Republic of Korea), maintained in Endothelial Cell Growth Medium-2 (EGM-2, PromoCell, Heidelberg, Germany), and used for assays at passages 3–8.

To construct the tubular liver tissue, HepG2 cells, HUVECs, and MSCs were expanded in culture dishes. HepG2 cells (JCRB1054, Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) were cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with 10% fetal bovine serum (FBS) (Biosera, Kansas City, MO, USA) and 1% penicillin-streptomycin solution (PS) (FUJIFILM Wako Pure Chemical). HUVECs (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium 2 (EGM2) (PromoCell), with a supplement kit and 1% PS. MSCs (SCRC-4000, ATCC, Manassas, VA, USA) were cultured in high-glucose DMEM, supplemented with 10% FBS, 1% non-essential amino acids solution (FUJIFILM Wako Pure Chemical), and 1% PS. In 2D cultures, all cell types were cultured independently in a mixture of equal volumes of the media for HepG2 cells and HUVECs in order to make conditions equivalent to those of the tubular liver tissue.